Schistosomiasis is a serious health problem and more than 200 million people are infected world wide. Like immunity to many helminth parasites, the immune response to Schistosoma mansoni is tightly linked to a robust Th2 response characterized by the expression of interleukin (IL)-4 by CD4+ T cells. The abrupt emergence of Th2 cells weeks after infection of the human or murine host coincides with the deposition of S. mansoni eggs. Intriguingly, injection of eggs alone induces a robust Th2 response even in the absence of infection or adjuvants. Despite the obvious importance of Th2 cells in the immune response to helminthic infections, research has been held back by the inability to follow parasite-specific CD4+ T cell responses. This is due to the fact that helminth-specific T cell receptors (TCR) have not been isolated to generate TCR transgenic mice. This situation is compounded by the fact that as of to date it is not possible to engineer antigen-transgenic helminth parasites that can be used in conjunction with existing TCR-transgenic mice. The availability of both pathogen- specific TCR transgenic mice and recombinant pathogens has been instrumental in studying immunity to many pathogens associated with Th1 immunity. Additionally, in contrast to many bacteria, protozoa and viruses, helminthic antigens are generally not known. One exception is IPSE/alpha-1, a basophil activating protein of S. mansoni eggs, whose function is conserved between men and mice. To follow helminth-specific Th2 responses by their signature, the expression of IL-4, we have previously developed IL-4 reporter mice (4get and KN2). We have demonstrated that expression of the IL-4 reporter is only induced when CD4+ T cells are exposed to both their cognate antigen and Th2 polarizing conditions. Consequently IL-4 expressing CD4+ T cells in helminth-infected mice are highly enriched for cells bearing TCRs specific for parasitic antigens. Here we will use IL-4 reporter mice to isolate helminth-specific Th2 cells generated in response to S. mansoni eggs and their antigens to establish Th2 cell lines and CD4+ T cell hybridomas. We have two independent aims. In Aim 1 we will functionally clone S. mansoni egg-specific Th2 cell hybridomas. We will isolate egg-specific Th2 cells either directly from immunized mice or use an adoptive transfer system to expand and reselect such cells in vivo. In Aim 2 we will functionally clone Th2 cells specific for the S. mansoni egg antigen IPSE/alpha-1. We will use a combination of antigen-specific immunization and selection to expand and reselect IPSE/alpha-1-specific Th2 cells for the generation of hybridomas. Our preliminary data with S. mansoni eggs, IPSE/alpha-1 reagents, IL-4 reporter mice and Th2 cell hybridomas demonstrate that we have acquired all required reagents and technical skill to pursue the proposed research. The identification of S. mansoni egg specific TCRs from cloned Th2 hybridomas is the first critical step in the generation of TCR transgenic mice. The generation of S. mansoni-specific hybridomas will be proof of principle for this new approach that can subsequently be used for other pathogens. Schistosomiasis is a serious health problem and more than 200 million people are infected world wide. Schistosoma mansoni is a multicellular helminth parasite that chronically infects men and mice. The adaptive immune response is in both men and mice tightly linked to a vigorous Th2 response that is initiated by and extensively directed against S. mansoni eggs. Despite the obvious importance of this disease, S. mansoni egg-specific Th2 cells and their T cell receptors (TCR) of have not been cloned. The isolation of pathogen-specific TCRs is a critical first step in the generation of antigen-specific TCR transgenic mice. Such pathogen- specific TCR transgenic mice have been instrumental in studying the immune response to many type 1 associated diseases caused by bacteria, viruses and protozoa. Here we will use a novel approach in which we employ interleukin (IL)-4 reporter mice to functionally isolate S. mansoni egg-specific Th2 cells by their signature cytokine, IL-4. Subsequently we will generate and clone T cell hybridomas to isolate the S. mansoni egg-specific TCRs. The work outlined in this application will be a critical step towards the generation of tools urgently needed to study schistosomiasis, other helminthic diseases and fundamental principles of Th2 immunity. [unreadable] [unreadable] [unreadable]